Subpicogram per milliliter determination of the tobacco-specific carcinogen metabolite 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol in human urine using liquid chromatography-tandem mass spectrometry

Abstract

Exposure to secondhand tobacco smoke (SHS) has been linked to increased risk for a number of diseases, including lung cancer. The tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is of particular interest due to its potency and its specificity in producing lung tumors in animals. The NNK metabolite 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) in urine is frequently used as a biomarker for exposure. Due to its long half-life (40-45 days), NNAL may provide a long-term, time-averaged measure of exposure. We developed a highly sensitive liquid chromatography-tandem mass spectrometry method for determination of NNAL in human urine. The method involves liquid-liquid extraction followed by conversion to the hexanoate ester derivative. This derivative facilitates separation from interfering urinary constituents by extraction and chromatography and enhances detection with electrospray ionization mass spectrometry. The lower limit of quantitation is 0.25 pg/mL for 5-mL urine specimens. Applications to studies of people with a range of different SHS exposure levels is described.

Figures

Figure 1

Formation and metabolism of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)

Figure 2

Product ion spectra of NNAL, NNAL acetate, and NNAL hexanoate.

Figure 3

Chromatograms of urine extracts containing NNAL, NNAL acetate, and NNAL hexanoate. Mobile phase (10 mM ammonium formate in methanol-water), column, and flow rate as described in experimental section. A was a linear gradient from 10% methanol to 70% methanol over 15 minutes; B was a linear gradient from 33% methanol to 100% methanol over 11 minutes.

Figure 4

Calibration curve for NNAL in urine. Six replicates at each concentration, linear regression, 1/x weighting. Lower panel is expanded scale for range of 0-1 pg/mL.

Figure 5

Chromatograms of non-smokers’ urine extracts. A, person with little or no SHS exposure; B, subject in study of SHS exposure in people with chronic obstructive pulmonary disease; C, college student after SHS exposure in a bar.

Figure 6

Distribution of NNAL concentrations in urine of 73 Research Subjects with chronic obstructive pulmonary disease. BLOQ = below limit of quantitation.

Figure 7

Concentrations of NNAL in urine of 8 college students prior to and after spending 6 hours in smoking-allowed bars. In the lower panel concentrations are normalized to creatinine to adjust for differences in urinary flow.